Cell mass capable of serving as a primitive organ-like structure comprised of a plurality of cell types of somatic origin

ABSTRACT

The present invention provides a method of producing a cell mass capable of serving as a primitive organ-like structure comprised of a plurality of somatic cell types of somatic origin, comprising: preparing cultures containing the plurality of types of somatic cells; mixing the plurality of types of somatic cell cultures followed by adding a Wnt signal activator to the mixed cell culture; subjecting the culture containing the Wnt signal activator to non-plate contact culturing over a predetermined time period; and replacing the medium of the culture cultured by the non-plate contact culturing with medium not containing Wnt signal activator and further culturing for a predetermined time period; wherein, at least one type of the plurality of somatic cells is maintained in an undifferentiated state.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/030,449, filed Sep. 18, 2013, which is a Divisional of U.S.application Ser. No. 12/306,649, which is the U.S. National Stageapplication of PCT/JP2007/063333, filed Jun. 27, 2007, which claimspriority from Japanese application JP 2006-176763, filed Jun. 27, 2006,the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 30, 2015, isnamed sequence.txt and is 5 KB.

TECHNICAL FIELD

The present invention relates to a method for producing a cell masscapable of serving as a primitive organ-like structure comprised of aplurality of cell types of somatic origin, and to a cell mass producedaccording to that method.

BACKGROUND ART

Due to the remarkable advances made in the field of medicine in recentyears, opportunities for saving lives are continuing to increase byeliminating the causes of diseases through, for example, systematictherapeutic techniques in the form of the surgical excision of cancer orliving donor transplant techniques for tissues and organs. However,there are cases in which patients suffer a considerable decrease in QOLaccompanying excision of an affected area. In addition, there arelimitations on treatment dependent upon living donor transplants due tosuch factors as a shortage of transplant donors and the occurrence ofrejection. If it were possible to regenerate a tissue or organ that hasbeen lost due to surgical treatment or an unforeseen accident, then itwould be possible to considerably improve the QOL of patients. Inaddition, regenerative medicine also makes it possible to resolve theproblems confronting living donor transplants. From this viewpoint, thedegree of expectations being placed on regenerative medicine is high.

Technologies in which regenerative medicine has been successful areprimarily related to comparatively simple tissue in terms of morphologyor function in the manner of artificial skin, artificial bone andartificial teeth. Reconstructed artificial skin and artificial bone isincorporated into cells enabling the providing of signals required fortissue construction. However, there have been limitations on therepertoire of differentiation of artificial skin and artificial bone byregenerative medicine techniques. For example, although allogenickeratinocytes or skin fibroblasts and the like differentiate intostructures in the form of the epidermis, are incorporated by surroundingorgans to eventually have a horny layer or basal layer having barrierproperties, there has been reported to be no derivation of secondaryderivatives such as hair follicles, sebaceous glands or sweat glands.

Body tissue normally contains both cells that are able to self-replicateand possess stem cell properties for maintaining tissue homeostasis bysending signals to differentiated cells or supplying differentiatedcells, and cells having properties of somatic cells that have alreadydifferentiated that receive various signals or commands from such cells,and is able to function through interaction between both of these typesof cells. In the case of vertebrates, for example, interaction betweenmesenchymal cells and epithelial cells is essential for nearly alltissue and organ formation. In the case of hair follicles, mesenchymalcells in the form of hair papilla cells are responsible for stemcell-like properties, while epithelial cells in the form ofkeratinocytes are equivalent to cells having somatic cell-likeproperties in the sense that they differentiate into hair shafts (hairitself).

The difficulty encountered when forming organs by regenerative medicinelies in reaching a state of coexistence between cells having stemcell-like properties maintained in an undifferentiated state and cellsthat have already differentiated as in actual body tissue. In the priorart, even if epithelial cells and mesenchymal cells were able to beco-cultured, they either both ended up differentiating or bothmaintained an undifferentiated state, thereby preventing thereproduction of the coexistence of undifferentiated cells anddifferentiated cells so as to mimic actual body tissue.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a cell mass capable ofserving as a primitive organ-like structure by combining and culturing aplurality of types of differentiated somatic cells.

The present application includes the following inventions:

(1) a method of producing a cell mass capable of serving a primitiveorgan-like structure comprised of a plurality of somatic cell types ofsomatic origin, comprising:

preparing cultures containing the plurality of types of somatic cells;

mixing the plurality of types of somatic cell cultures followed byadding a Wnt signal activator to the mixed cell culture;

subjecting the culture containing the Wnt signal activator to non-platecontact culturing over a predetermined time period; and

replacing the medium of the culture cultured by the non-plate contactculturing with medium not containing Wnt signal activator and furtherculturing for a predetermined time period; wherein,

at least one type of the plurality of somatic cells is maintained in anundifferentiated state;

(2) the method of (1), wherein the plurality of types of somatic cellsis comprised of combinations of epithelial somatic cells and mesenchymalsomatic cells, and the mesenchymal somatic cells are maintained in anundifferentiated state;

(3) the method of (2), wherein the epithelial somatic cells arekeratinocytes, and the mesenchymal somatic cells are hair papilla cells;

(4) the method of (3), wherein hair follicles having a hair follicleinducing function are formed from the cell mass;

(5) the method of any of (1) to (4), wherein the Wnt signal activator is6-bromoindirubin-3′-oxime (BIO);

(6) the method of any of (1) to (5), wherein the non-plate contactculturing method is the hanging drop method;

(7) a cell mass capable of serving as a primitive organ-like structurecomprised of a plurality of somatic cell types of somatic origin,produced by a method comprising:

preparing cultures containing the plurality of types of somatic cells;

mixing the plurality of types of somatic cell cultures followed byadding a Wnt signal activator to the mixed cell culture;

subjecting the culture containing the Wnt signal activator to non-platecontact culturing over a predetermined time period; and

replacing the medium of the culture cultured by the non-plate contactculturing with medium not containing Wnt signal activator and furtherculturing for a predetermined time period; wherein,

at least one type of the plurality of somatic cells is maintained in anundifferentiated state;

(8) the cell mass of (7), wherein the plurality of types of somaticcells is comprised of combinations of epithelial somatic cells andmesenchymal somatic cells, and the mesenchymal somatic cells aremaintained in an undifferentiated state;

(9) the cell mass of (8), wherein the epithelial somatic cells arekeratinocytes, and the mesenchymal somatic cells are hair papilla cells;

(10) the cell mass of (9), wherein hair follicles having a hair follicleinducing function are formed from the cell mass;

(11) the cell mass of any of (7) to (10), wherein the Wnt signalactivator is 6-bromoindirubin-3′-oxime (BIO);

(12) the cell mass of any of (7) to (11), wherein the non-plate contactculturing method is the hanging drop method;

(13) a method for screening for drugs having a hair growth effect byapplying a candidate drug to the cell mass of any of (9) to (12); and,

(14) the method of (13), which uses as an indicator an expressed amountof one or a plurality of genes selected from the group consisting ofc-myc, BMP4 and IGFBP3 by the cell mass.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an immunostaining diagram (1) of a cell mass producedaccording to the method of the present invention showing staining of DPcells and NHEK cells;

FIG. 2 is an immunostaining diagram (2) of a cell mass producedaccording to the method of the present invention showing localization ofhair papilla cells in the cell mass;

FIG. 3 is an immunostaining diagram (3) of a cell mass producedaccording to the method of the present invention showing cellproliferation in the cell mass;

FIG. 4 is an immunostaining diagram (4) of a cell mass producedaccording to the method of the present invention showing staining ofβ-catechin and NHEK cells;

FIG. 5 is an immunostaining diagram (5) of a cell mass producedaccording to the method of the present invention showing expression ofhair keratin in the cell mass;

FIG. 6 is an immunostaining diagram (6) of a cell mass producedaccording to the method of the present invention showing expression ofhair buds in the cell mass;

FIG. 7 shows the expression of various genes in RNA originating in acell mass of the present invention (1);

FIG. 8 shows the expression of Wnt3A gene in RNA originating in a cellmass of the present invention;

FIG. 9 shows the expression of Wnt10B gene in RNA originating in a cellmass of the present invention; and

FIG. 10 shows the expression of various genes in RNA originating in acell mass of the present invention (2).

BEST MODE FOR CARRYING OUT THE INVENTION

According to the present invention, a cell mass can be provided that iscapable as serving as a primitive organ-like structure comprised of aplurality of types of somatic cell types.

Somatic cells as referred to in the present invention refer to cellsthat have reached differentiation into cells that compose various organsof the body, and refer to cells that are the opposite ofundifferentiated stem cells. The present invention is characterized bythe use of two or more somatic cells, and preferably consists of variouscombinations thereof, such as a combination of an epithelial cell lineand mesenchymal cells, a combination of endothelial cells andmesenchymal cells, or a combination of epithelial cells and mesenchymalcells.

There are no particular limitations on organs capable of being formed bythe cell mass as claimed in the present invention, examples of whichinclude various organs such as hair follicle, lung, kidney, liver,pancreas, spleen, heart, gallbladder, small intestine, colon, largeintestine, joint, bone, tooth, blood vessel, lymph duct, cornea,cartilage, olfactory organ or auditory organ.

For example, in the case of desiring to form a hair follicle,keratinocytes of the head region are used for the epithelial cell linewhile hair papilla cells are used for the mesenchymal cells. The“epithelial cells” referred to here refer to cells that compose themajority of the epidermis or epithelium of skin, and arise from a singlelayer of basal cells in contact with the dermis. The epithelial cellsmay be epithelial cells originating in newborns (or fetuses), cellsoriginating in mature skin such as the epidermis of dormant skin or theepidermis of growing skin, or a culture of cells having the morphologyof keratinocytes. Such cells can be prepared from the skin of a desireddonor animal according to methods known among persons with ordinaryskill in the art. “Hair papilla cells” refer to cells located at thelowermost portion of hair follicles as mesenchymal cells that fulfillthe role of a so-called control tower by sending activating signals tohair follicle epithelial stem cells for auto-regeneration of hairfollicles. “Hair papilla cells” can be prepared by, for example,preparing a cell suspension by treating a dermal tissue fraction,obtained by removing epidermal tissue from skin tissue, with collagen,and then placing the cell suspension in frozen storage to destroy thehair follicle epithelial cells.

In addition, preparation can be carried out in the following manner forreconstruction of an olfactory organ, for example. First, a tissue siteis excised at which is present a group of olfactory epithelial cells ofa mammal such as a mouse. The tissue is then digested by treating withcollagenase and separated by centrifugation to prepare a precipitatedcell group. Then, the cell suspension is placed in a cell culture dishcoated with collagen 1 or 4, and the suspension is rapidly aspirated inabout 5 minutes to allow the highly adhesive olfactory epithelial cellsto preferentially adhere. Epithelial cells remaining in the same dishcan be transferred to a cell culture. Moreover, the majority of theremaining cells are epithelial cells. Although mesenchymal cells arepresent in the remaining aspirated suspension, by placing the suspensionin a culture dish coated with fibronectin or gelatin and allowing tostand undisturbed for 4 hours or more in a suitable atmosphere such asthat at 37° and 95% CO₂, the mesenchymal cells can be applied to cellculturing. The epithelial and mesenchymal cells prepared as describedabove can be used to reconstruct a new olfactory organ using a proceduresimilar to that used to reconstruct hair follicle organs.

Moreover, preparation can be carried out in the following manner forreconstruction of a renal glomerulus, for example. First, a site wheretissue is present in the kidney of a mammal such as a mouse is excised.The tissue is digested by treating with collagenase and the separated bycentrifugation followed by removing coarse matter with a 100 μm meshfilter. Then, glomerulus is collected in the mesh with a 40 μm meshfilter and a cell suspension is obtained by treating with trypsin. Thecell suspension is placed in a cell culture dish coated with collagen 1or 4 to preferentially adhere epithelial cells. As a result of thistreatment, the majority of the cells are epithelial cells originating inthe glomerulus.

On the other hand, in the case of kidney, mesenchymal cells present atthe time of organogenesis are destroyed in the body. Thus, in thepresent invention, the following procedure can be employed to re-induceepithelial-mesenchymal interaction required for tissue reconstruction.The renal cells described above are used for the epithelial cells.Moreover, hair papilla cells or bone marrow mesenchymal cells of thesame category are used as a substitute for the lost mesenchymal cells.Epithelial and mesenchymal cells as described above can be used toconstruct a new renal organ by using a technique similar to that used toreconstruct hair follicle organs.

Various mammals can be used without limitation as the origin of thecells as claimed in the present invention corresponding to the purposethereof, examples of which include chimpanzees, other primates, domesticanimals such as dogs or cats, farm animals such as cows, pigs, horses,sheep or goats, laboratory animals such as rabbits, rats, mice or guineapigs, and more preferably nude mice, SCID mice or nude rats. Inaddition, although combinations thereof may be homogeneous combinationsor heterogeneous combinations, homogeneous combinations are preferable.

There are no particular limitations on culture media effective forculturing the somatic cells as claimed in the present invention, andmedia commonly used for cell culturing can be used. For example, in thecase of culturing mesenchymal cells, serum-containing media such asDMEM, MEM, F12 or Chang medium can be used preferably. In this case,serum concentration is 0 to 30% and preferably 10%, and basic fibroblastgrowth factor (bFGF), epidermal growth factor (EGF) and 2 mM L-glutamineare added as essential factors.

In the case of epithelial cells, serum-free medium optimized forkeratinocytes, such as Epilife (registered trademark) and HuMedica(registered trademark) (both available from Kurabo) or Invitrogen SFM(available from Invitrogen), is preferable. Although keratinocytes canbe prepared using the Green method depending on the case (Cell 1975November; 6(13): 331-43, Serial cultivation of strain of human epidermalkeratinocytes: the formation of keratinizing colonies from single cells,Rheinwalf J. G., Green H.), in this case, serum-containing medium usedin the mesenchymal cell system described above can be used for theculturing thereof. Furthermore, examples of antibiotics that can be usedfor culturing both epithelial cells and mesenchymal cells includepenicillin G, kanamycin, streptomycin and amphotericin B.

The present invention is characterized by the addition of a Wnt signalactivator to a mixture of the plurality of somatic cells as describedabove followed by culturing thereof. Wnt signaling refers to a series ofactions that demonstrate the function of transcription factors bypromoting nuclear migration of β-catechin. These signals originate fromcellular interaction that includes, for example, a series of processesin which a protein referred to as Wnt3A secreted from certain cellsfurther acts on other cells causing nuclear migration of intracellular βcatechin which acts as a transcription factor. This series of processesgive rise to the initial phenomenon of organ construction in the exampleof epithelial-mesenchymal interaction. Wnt signaling is known to controlcell proliferation and differentiation, organ formation and various cellfunctions such as cell migration during initial development. AlthoughWnt signaling is used when culturing ES cells for the purpose ofinhibiting differentiation due to their function of maintaining anundifferentiated state (see, for example, Noboru Sato et al., NatureMedicine, Vol. 10, No. 1, January 2004), their utilization and effectsduring culturing of somatic cells are completely unknown.

There are no particular limitations on the Wnt signal activator, and anyactivator can be used provided it demonstrates inhibitory activity onglycogen synthase kinase-3 (GSK-3), examples of which include abis-indolo(indirubin) compound, (BIO)((2′Z,3′E)-6-bromoindirubin-3′-oxime), its acetoxime analog,BIO-acetoxime (2′Z,3′E)-6-bromoindirubin-3′-acetoxime), thiadiazolidine(TDZD) analog (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione) andoxothiadiazolidine-3-thione analog(2,4-dibenzyl-5-oxothiazolidine-3-thione), a thienyl α-chloromethylketone compound (2-chloro-1-(4,4-dibromo-thiophen-2-yl)-ethanone), aphenyl α-bromomethyl ketone compound (α-4-dibromoacetophenone),thiazol-containing urea compound(N-4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea) and GSK-313peptide inhibitors such as H-KEAPPAPPQSpP—NH₂.

There are no particular limitations on the amount of Wnt signalactivator added, and the amount added is required to be an amount atwhich Wnt signal activation, or in other words, inhibition of GSK-3, isdemonstrated and cell proliferation is not interrupted, is dependent onthe type of cells to be grown, and is suitably determined by a personwith ordinary skill in the art. For example, in the case of using BIOfor the Wnt signal activator in hair papilla cells, although an amountadded of, for example, about 0.1 to 10 μM is suitable, the amount addedis naturally not limited thereto.

Another characteristic of the present invention is the subjecting of themixture of the plurality of somatic cells, to which Wnt signal activatorhas been added, to non-plate contact culturing. Non-plate contactculturing refers to a method of culturing cells on an interface having aspherical surface so as not to allow adhesion of plate-adhering cells.An example of non-plate contact culturing is a hanging drop method(Keller, G. M. et al., Curr. Opin. Cell Biol., 7, 862-869 (1995)). Thehanging drop method refers to adhering a drop (typically about 25 to 35μL) of culture medium containing cultured cells onto the inside of theupper lid of a culture dish, carefully closing the lid so that theculture medium does not drop or run down, and culturing cells within theculture medium to be cultured in the form of an inverted drop due tosurface tension. As a result of culturing in this manner, the effects onthe cells attributable to contact with a flat surface as in the case ofplate culturing can be minimized. Other examples of non-plate contactculturing methods include a formation method utilizing a semi-sphericalcell culture dish that has been surface-treated in advance to preventcell adhesion (for example, “Spheroid” commercially available fromSumitomo Bakelite) (referred to as the spheroid formation method), and asuspension method in which cells are aggregated in a suspended state byculturing in a nitrocellulose medium.

Although the temperature and duration of non-plate contact culturingvaries dependent on the type of cells handled and the number of timesthey have been subcultured, culturing is carried out, for example, at37° C. for 1 to 10 days and preferably for 3 to 7 days, and the mediumis suitably replaced with the same medium as necessary.

Finally, the cells are further subjected to non-plate contact culturingin the absence of Wnt signaling by replacing the above medium withculture medium not containing Wnt signal activator. In this case aswell, although the temperature and duration of culturing variesdependent on the type of cells handled and the number of times they havebeen subcultured, culturing is carried out, for example, at 37° C. for 1to 10 days and preferably for 3 to 7 days, and the medium is suitablyreplaced with the same medium as necessary.

According to the present invention, a cell mass useful for, for example,hair follicle transplant, various organ transplants and partialtransplants of microorgans comprising organs although not completeorgans themselves, such as cornea, renal glomerulus, olfactoryepithelial tissue or a cartilage site, can be efficiently andartificially produced. In addition, by using an artificially producedcell mass for drug evaluations, the action of a drug on organs of thebody can be evaluated more accurately. In addition, transplant of thecell mass into immunosuppressed animals such as nude mice or SCID micemakes it possible to produce more complete organ-like tissue, and byectopically producing human hair follicle or mouse kidney, for example,animal models can be prepared enabling evaluation of new drugs andevaluation of gene function. In particular, by inserting or altering agene in cells when producing the cell mass, reconstructed organs havinga reporter can be easily produced, thereby making it possible toaccurately determine and investigate molecular behavior in the body atthe tissue and organ levels.

In the cell mass according to the present invention, the epithelialcells are keratinocytes and the mesenchymal cells are hair papillacells, and in the case of forming hair follicles having a hairfollicle-inducing function, the cell mass can be used to evaluate drugsdemonstrating a hair growth effect, and can be used, for example, byscreening for such drugs. For example, in the case of applying acandidate drug of a hair growth drug to this cell mass and the growth ofthe cell mass is remarkable in comparison with a control, the drug canbe assessed to have a hair growth effect, or can be assessed for thepresence or absence of a hair growth effect by using increasedexpression of a gene that promotes hair growth, such as c-myc, as anindicator or by using inhibition of the expression of a gene thatinhibits hair growth, such as BMP4 or IGFBP3, as an indicator.

EXAMPLES

Hair Papilla Cells

Human hair papilla cells were prepared from scalp tissue provided by adonor. After removing the dermal tissue, hair follicle sites present infatty tissue were collected using forceps and ophthalmic scissors whileviewing under a stereo microscope. The collected hair follicles weretransferred to culture medium containing antibiotics, and sites of hairpapilla cells were visually isolated and collected under the samemicroscope. The isolated hair papilla cells were cultured in the mediumfor one week or more at 37° C. and 95% CO₂ in a 10 cm round dish (TRP),after which they were submitted for experimental use. The medium usedcomprised Advanced DMEM (Invitrogen), 15% fetal bovine serum, 20 ng/mlof bFGF, 10 ng/ml of EGF, 2 mM L-glutamine, a mixture of penicillin,streptomycin and amphotericin (100-fold dilution), and 3.5 μl/50 ml ofβ-mercaptoethanol. The cells were suitably subcultured under the sameconditions. During subculturing, the cells were separated with 0.5%trypsin/EDTA solution, transferred to a fresh dish and then sub-culturedin fresh medium of the same composition.

In the case of adding BIO (Calbiochem), the BIO was dissolved to 10 mMwith dimethylsulfoxide (DMSO) and added to a concentration of 0.1 to 5μM.

Keratinocytes

Keratinocytes from human normal neonatal foreskin were prepared in themanner described below. Foreskin tissue collected from a donor wastreated overnight at 4° C. in PBS(−) in the presence of dispase enzyme.Only the epidermis was able to be collected in the form of a sheet as aresult of this treatment, and this was digested with 0.25% trypsinenzyme using the same method as during subculturing, after which thetissue and medium were transferred to a collagen-treated culture dishand cultured. Humedia KG2 (Kurabo) was used for the medium. The cellswere suitably subcultured under the same conditions. Duringsubculturing, the cells were separated with 0.5% trypsin/EDTA solution,transferred to a fresh dish and then sub-cultured in fresh medium of thesame composition. Cells collected and cultured in the manner describedabove were used in subsequent experimentation.

In the case of adding BIO (Calbiochem), the BIO was dissolved to 10 mMwith dimethylsulfoxide (DMSO) and added to a concentration of 0.1 to 5μM.

Hanging Drop Culturing

Medium Composition:

30% Advanced DMEM (Invitrogen), 2 mM L-glutamine, a mixture ofpenicillin and streptomycin (Invitrogen, 100-fold dilution) and 3.5μl/50 ml of β-mercaptoethanol were mixed with Humedia KG-2 (Kurabo) at aratio of 1:1.

In the case of adding BIO (Calbiochem), the BIO was dissolved to 10 mMwith dimethylsulfoxide (DMSO) and added to a concentration of 0.1 to 5μM.

Preparation Method:

Hair papilla cells were prepared to P3 or more (with P1 indicating oneround of subculturing) in the presence or absence of BIO loading, whilekeratinocytes were prepared up to and including P3.

Each of the cells was diluted with each medium following trypsintreatment so as to obtain 1×10⁵ cells.

In the case of adding BIO (Calbiochem), the BIO was dissolved to 10 mMwith dimethylsulfoxide (DMSO) and added to a concentration of 0.1 to 5μM.

25 to 35 μl of each culture medium was dispensed onto the inside of theupper lid of a square culture dish measuring 10 cm on a side and mixedto prepare dome-shaped droplets of a suitable size.

After carefully closing the lid, the cells were cultured at 37° C. in a95% CO₂ atmosphere while ensuring that the culture medium did not dropdown.

Three days later, the medium was replaced with the same medium as thatused above.

After culturing for an additional four days, a portion of the medium wassubjected to RT-PCR analysis (1) as described below. All of theremaining media was replaced with medium under conditions of the absenceof BIO.

The media was replaced in the same manner on the third day, and cellmasses that formed on day 7 were collected and subjected toimmunostaining as described below.

Immunohistofluorescent Staining:

Each of the cell masses obtained with the hanging drop method were fixedfor 5 minutes with 0.1% paraformaldehyde and submitted for staining. Inaddition, a portion of the cell masses were embedded in OCT solutionwithout fixing and prepared into frozen sections with a cryostatfollowed by immunohistostaining.

Blocking was carried out for 6 hours with PBS(−) containing 13% bovineserum albumin (BSA).

Each of the primary antibodies indicated in the following table werediluted by 1/200 with the aforementioned PBS(−) containing 13% bovineserum albumin (BSA), added to each sample and allowed to react for 4hours at 4° C.

The cells were washed three times with PBS(−) containing 0.02% Tween 20.

Fluorescent secondary antibody (see Excel file) diluted by 1/200 withthe aforementioned PBS(−) containing 13% bovine serum albumin (BSA) wasadded to each sample. In addition, the nuclei of the cells were stainedblue with DAPI (4′-6-diamidino-2-phenylindole).

The cells were observed with a fluorescent microscope (Olympus).

TABLE 1 Target Protein Manufacturer Description Primary CD49f SerotecRat anti-human/ antibody mouse CD49f Vimentin YLEM Monoclonal β-catechinBecton Dickinson Monoclonal Name Manufacturer Description Secondary Goatanti-rat IgG Jackson Red antibody TEXAX-RED ImmunoResearch conjugateRabbit anti-mouse IgG Invitrogen Yellow ALEXAFLUOR 488

Here, since monoclonal antibody to vimentin specifically stains hairpapilla cells and Cd49f polyclonal antibody specifically stainsepidermal cells, application of vimentin and CD49f antibodies toimmunocytostaining makes it possible to evaluate the attributes of cellspresent within the cell masses formed in this experimental system.

FIG. 1 shows black-and-white photographs of the results of themicroscopic observations described above. In the color version of thisdiagram, green coloring by vimentin antibody and red coloring by CD49fantibody can be distinguished, and each were able to be determined to behair papilla cells and keratinocytes, respectively. In addition, asshown in FIG. 2, hair papilla cells were confirmed to be aggregated inthe central portion of the cell mass. As a result of these diagrams, thecells were observed to be regularly arranged after having aggregated,and epithelial cells and mesenchymal cells were determined to bearranged in an orderly manner by this experimental system. After thecell masses formed following the addition of BIO, red-stainingkeratinocytes were determined to be growing in a regular manner in theform of branches within cell masses cultured in the absence of BIO.Moreover, as shown in FIG. 3, in the case of forming a cell mass byadding BIO, a central portion where hair papilla had aggregated and anend portion of the cell mass that extended in the form of a branch werefound to be positive for the proliferation marker, K167. On the basis ofthese results, although normal aggregated somatic cells usually enter adormant state without growing, it was unexpectedly found thatproliferation of cells occurred in the cell masses due to the additionof BIO.

In addition, β-catechin monoclonal antibody was used to mark stemcell-like properties. Since β-catechin is present in small amounts nearthe cell membrane of normal somatic cells, it can only be slightlydetected by ordinary fluorescent immunostaining. However, in cellshaving stem cell-like properties, β-catechin acts as a transcriptionfactor within the nucleus and is present in greater amounts. Moreover,since β-catechin accumulates in the nucleus, it can be confirmed to bestrongly localized even by fluorescent immunostaining. In thisexperiment as well, since β-catechin was strongly stained at somespecific sites of the cell mass and was confirmed to be accumulated atthose sites, staining with β-catechin antibody can be said to besuitable for detection of cells having stem cell-like properties in thecell mass.

FIG. 4 is a black-and-white photograph showing the results ofmicroscopic observation using the aforementioned β-catechin monoclonalantibody staining. In the color version of this diagram, sites stainedwith β-catechin antibody are shown in green, while keratinocytes areshown in red. β-catechin-positive cells were observed at sites emittingan intense green light, thus indicating the cells to have stem cell-likeproperties. What should be noted here is the presence of portions atwhich an intense yellow light is observed in the centers of sphericalsites of the cell mass. These sites indicate β-catechin-positivekeratinocytes. β-catechin-positive cells can be said to be cells havingstem cell-like properties as previously described. On the other hand,there are no yellow sites at sites in keratinocytes extending from thecell mass in the form of branches. In other words, cells having stemcell-like properties and cells having normal somatic cell-likeproperties (keratinocytes) can be observed to both be present. Thisstate is unable to be observed during ordinary culturing, andepithelial-mesenchymal interaction is reproduced as if it were presentin vivo, thus indicating that undifferentiated and differentiated cellgroups are growing by autonomously forming a cell mass.

Undifferentiated keratinocytes as referred to here indicate cells havinga stem cell-like ability to constantly create keratinocytes that formhair in the manner of hair matrix cells. On the other hand,differentiated keratinocytes are limited as to the number of times theyproliferate, and indicate so-called cells for which their cell fate isdetermined as they become hair. At the same time, although cellssurrounding green spheres in the color version of FIG. 4 are thought tobe hair papilla cells, the fact that hair papilla cells in theenvironment of this cell mass are β-catechin-positive cells is importantin the same manner that Wnt signaling is important during follicleformation and at the start of growth in vivo.

Antibody to cytokeratin 14 (K14) expressed in epidermal keratinocytesand outer root sheath cells is used for the purpose of confirming thatthese cells have not differentiated to hair roots (red). Since AE13monoclonal antibody, which specifically reacts to hair keratin (Lynch etal., J. Cell Biol., 103, 2593, 1986), recognizes 44K/46K acidic hairkeratin dimer, it is used to mark differentiation into hair roots(green). Hair keratin is a hair follicle-specific structural proteinthat is essentially absent in normal epidermal keratinocytes. FIG. 5 isa black-and-white photograph showing the results of microscopicobservation thereof. In the color version of this diagram, althoughsites stained by AE13 antibody are not observed in cell masses producedwith hair papilla cells and epidermal keratinocytes, when stimulatedwith BIO, staining is observed in some cell masses. In cell massesprepared with hair papilla cells and outer root sheath cells, sitesstained by AE13 antibody can be confirmed in cases of the absence of BIOstimulation, and staining by AE13 antibody is considerably enhanced byBIO stimulation. On the other hand, since sites stained by both K14antibody (red) and AE13 antibody (green) (yellow sites) are not observedin any of the cell masses, a change from undifferentiated cellsexpressing cytokeratin 14 to cells producing hair keratin is thought tohave occurred.

Ber-EP4 antibody to human epithelial antigen specifically expressed inhair buds during the hair growth stage (Ogawa, et al., Exp. Dermatol.13, 401, 2004) is used to mark hair buds during hair growth (green).FIG. 6 is a black-and-white photograph showing the results ofmicroscopic observation thereof. In the color version of this diagram,immunostaining (green) by Ber-EP4 antibody is clearly observed in thecase of having stimulated cell masses prepared with hair papilla cellsand outer root sheath cells with BIO. Since sites (yellow) stained byboth K14 antibody (red) and Ber-EP4 antibody (green) are not observed, achange from undifferentiated cells expressing cytokeratin 14 to cellsdifferentiated to hair buds expressing Ber-EP4 is thought to haveoccurred.

RT-PCR Analysis (1)

RNA was extracted from each sample using Trizol (Invitrogen). Anequivalent of 200 ng of each RNA was reverse transcribed to cDNA with areaction system consisting of 50 μl of RevTraACE (Toyobo). 1 μl of theresulting samples was applied to a PCR reaction in a 50 μl system usingthe primers listed below. KOD-DASH (Toyobo) was used for the enzyme, andthe reaction protocol consisted of 60 seconds at 95° C., 40 cycles of 30seconds at 95° C., 15 seconds at 63° C. and 30 seconds at 72° C.,followed by 60 seconds at 72° C.

10 μl of the reaction solution was electrophoresed at 100 V in 2%agarose gel (Cosmobio)/TAE buffer. A 100 by ladder marker (Toyobo) wasused for the DNA size marker.

The primers used are listed in the following table, and the PCR resultsare shown in FIG. 7.

TABLE 2 Sense primer Anti-sense primer (forward chain)  (reverse chain)Lef-1 gctatcaaccagattcttggcagaagg cagctgtcattcttggacctgtacctg(SEQ ID NO. 1) (SEQ ID NO. 2) SHH ctacgagtccaaggcacatatccactgtccaggaaagtgaggaagtcgctgtag (SEQ ID NO. 3) (SEQ ID NO. 4) STAT-3aagaccggcgtccagttcactactaaag ggtcaagtgtttgaattctgcagagagg (SEQ ID NO. 5)(SEQ ID NO. 6) Versican tccaagttatgttggtgcactttgtgagtcaaacatcttgtcattgaggcctatcc (SEQ ID NO. 7) (SEQ ID NO. 8) TWIST-1ggccaggtacatcgacttcctctac tctccttctctggaaacaatgacatc (SEQ ID NO. 9)(SEQ ID NO. 10) β-actin cgcgagaagatgacccagatcatg ccacaggactccatgcccacg(SEQ ID NO. 11) (SEQ ID NO. 12)

β-actin was used as a control for the entire experiment.

Lef-1 is a downstream gene of Wnt signaling, and as shown in FIG. 7, wasdetected in this experimental system both in the presence and absence ofBIO. The observation of expression of Lef-1 gene means that Wnt signalsare acting on the cells. Thus, Wnt signaling can be understood to befunctioning by addition of BIO in this experimental system.

SHH (Sonic Hedge Hog) is a gene that is involved during tissueformation, and as shown in FIG. 7, was not detected in this experimentalsystem either in the presence or absence of BIO. SHH is a gene that istranscribed and expressed in response to Wnt and Lef-1 signals.Expression of this gene indicates that Wnt signaling is acting on thecells and that linked gene expression such as that which occurs in vivois occurring. At the same time, SHH is a protein responsible forepithelial-mesenchymal intercellular signaling, and simultaneouslysuggests the occurrence of epithelial-mesenchymal interaction.

STAT-3 (Signal Transducer and Activator of Transcription 3) is a generelated to induction of hair follicles, and as shown in FIG. 7, wasdetected prominently in the presence of BIO as compared with in theabsence thereof. STAT-3 is a so-called intracellular signal transductionprotein involved in self-proliferation of stem cells. Acceleration ofthis gene is an essential factor for self-proliferation and maintaininguniversality in embryonic stem cells, and is one of the markers used toindicate stem cell-like properties thereof. At the same time, as isdescribed in the paper by Sano, S. et al. (Nature Med. 11: 43-49, 2005,“Stat3 links activated keratinocytes and immunocytes required fordevelopment of psoriasis in a novel transgenic mouse model”), since thisis also an extremely important gene for transition to the hair growthstage, it is suggested that acceleration of transcription of STAT-3 inthis experimental system is a normal stage during subsequent growth intohair follicles. At the same time, the STAT-3 signal itself is notdirectly affected by BIO (Sato, N. et al., Nat. Med. 2004, January;10(1):55-63, Epub 2003 Dec. 21, “Maintenance of pluripotency in humanand mouse embryonic stem cells through activation of Wnt signaling by apharmacological GSK-3 specific inhibitor”). In other words, accelerationof STAT-3 during addition of BIO indicates that a stem cell maintenancesystem is created within cells by epithelial-mesenchymal interaction.

TWIST-1 is marker for embryonic mesenchymal stem cells, and suggeststhat hair papilla cells have extremely high stem cell-like properties.Since expression of TWIST-1 in somatic cells is limited to an extremelyrare type of cells containing bone marrow mesenchymal stem cells, hairpapilla cells can be said to be cells having a high degree of stemcell-like properties despite being somatic cells. In the RT-PCRexperiment of FIG. 7, RT-PCR is carried out using equal amounts of wholeRNA for the templates. The reason for expression appearing to decreasein the presence of BIO is due to a relative decrease in the number ofhair papilla cells among the total number of cells as can be observedfrom FIG. 1 and FIG. 2. In conclusion, even when considering variationsin expressed amounts, the continued presence of cells expressing TWIST-1gene suggests that hair papilla cells capable of having stem cell-likeabilities are continuing to be present in this experimental system.

Versican gene is a hair growth marker known to be strongly expressed inhair papilla cells during induction of hair growth (Kishimoto, et al.,Proc. Natl. Acad. Sci. USA (1999), pp. 7336-7341). In the RT-PCRexperiment of FIG. 7, the reason for expression appearing to decrease inthe presence of BIO is due to a relative decrease in the number of hairpapilla cells among the total number of cells as can be observed in FIG.1 and FIG. 2. In conclusion, even when considering variations inexpressed amounts, the continued presence of cells expressing Versicangene suggests that hair papilla cells capable of inducing hair growthare continuing to be present in this experimental system.

Based on the changes in the expression levels of each gene,undifferentiation of epithelial and mesenchymal cells is maintained dueto the direct effects of BIO within the cell mass, and an actionmimicking the epithelial-mesenchymal action induced by Wnt signaling canbe said to have occurred. In addition, since the appearance of stemcell-like properties unrelated to Wnt signaling are observed as withSTAT-3, the effect of BIO contributes to the creation of a cell masshaving autonomic cell interaction and the ability to proliferate.

Wnt3A RT-PCR

Hair papilla cells were prepared to P3 (with P1 indicating one round ofsubculturing) in the presence of BIO loading, while keratinocytes wereprepared up to and including P3.

Each of the cells was diluted with each medium following trypsintreatment so as to obtain 3×10³ cells.

BIO (Calbiochem) was dissolved to 10 mM with dimethylsulfoxide (DMSO)and added to a concentration of 0.1 to 5 μM.

25 to 35 μl of each culture medium was dispensed onto the inside of theupper lid of a square culture dish measuring 10 cm on a side and mixedto prepare dome-shaped droplets of a suitable size.

After carefully closing the lid, the cells were cultured at 37° C. in a95% CO₂ atmosphere while ensuring that the culture medium did not dropdown.

Three days later, the medium was replaced with the same medium as thatused above. The samples were collected on the 7th day and submitted forRT-PCR analysis (2) as described below (“0 day” in FIG. 8). The mediumused for the same remaining cells was replaced with medium underconditions of the absence of BIO to induce differentiation, after whichthe samples collected on the 3rd day were submitted for RT-PCR analysis(2) as described below (“3 days” in FIG. 8).

RT-PCR Analysis (2)

RNA was extracted from the samples using Trizol (Invitrogen). Anequivalent of 200 ng of each RNA was reverse transcribed to cDNA with areaction system consisting of 50 μl of RevTraACE (Toyobo). 1 μl of theresulting samples was applied to a PCR reaction in a 50 μl system usingthe primers listed below. KOD-DASH (Toyobo) was used for the enzyme, andthe reaction protocol consisted of 2 minutes at 94° C., 32 cycles of 30seconds at 94° C., 10 seconds at 63° C. and 30 seconds at 72° C.,followed by 2 minutes at 72° C.

Sense primer (forward chain): (SEQ ID NO. 13) caggaactacgtggagatcatg Anti-sense primer (reverse chain): (SEQ ID NO. 14)ccatcccaccaaaactcgatgtc 

10 μl of the reaction solution was electrophoresed at 100 V in 2%agarose gel (Cosmobio)/TAE buffer. Target bands were detected aftervisualizing with ethidium bromide.

The results are shown in FIG. 8. The arrow in the drawing indicates aWnt3A band. The expression itself of Wnt3A is rare, is mainly expressedby epithelial cells, and is known to be a gene involved in organogenesisand epithelial cell-mesenchymal cell interaction. As shown in thedrawing, in contrast to expression of Wnt3A not being observed at all incells in which Wnt signaling was activated by culturing in the presenceof BIO, expression of Wnt3A was observed in cells in which celldifferentiation was subsequently induced by removal of BIO. Accordingly,in this experimental system, keratinocyte differentiation, and what ismore, autonomous organogenesis were clearly determined to be induced byculturing in the presence of BIO followed by culturing after removingBIO.

Wnt10B RT-PCR

Hair papilla cells were prepared to P3 (with P1 indicating one round ofsubculturing) in the absence of BIO loading, while outer root sheathcells were prepared up to and including P3.

Each of the cells was diluted with each medium following trypsintreatment so as to obtain 3×10³ cells.

BIO (Calbiochem) was dissolved to 10 mM with dimethylsulfoxide (DMSO)and added to a concentration of 0.1 to 5 μM.

25 to 35 μl of each culture medium was dispensed onto the inside of theupper lid of a square culture dish measuring 10 cm on a side and mixedto prepare dome-shaped droplets of a suitable size.

After carefully closing the lid, the cells were cultured at 37° C. in a95% CO₂ atmosphere while ensuring that the culture medium did not dropdown.

Three days later, the medium was replaced with the same medium as thatused above. The samples were collected on the 7th day and submitted forRT-PCR analysis (3) as described below (“day 7” in FIG. 9). The mediumused for the same remaining cells was replaced with medium without BIOto induce differentiation, after which samples collected after 10 and 14days were submitted for RT-PCR analysis (3) as described below (“day 10”and “day 14” in FIG. 9).

RT-PCR Analysis (3)

RNA was extracted from the samples using Trizol (Invitrogen). Anequivalent of 200 ng of each RNA was reverse transcribed to cDNA with areaction system consisting of 50 μl of RevTraACE (Toyobo). 4 μl of theresulting samples were applied to a quantitative PCR system (LightCyclerSystem, Roche) in a 20 μl system using the primers listed below. Thereaction protocol of LightCycler FastStart DNA Master SYBR Green (Roche)(consisting of 40 cycles of 10 seconds at 95° C., 10 seconds at 63° C.and 15 seconds at 72° C.) was used.

Sense primer (forward chain): (SEQ ID NO. 15)gaagttctctcgggatttcttggatcc  Anti-sense primer (reverse chain):(SEQ ID NO. 16) cggttgtgggtatcaatgaagatgg 

The results are shown in FIG. 9. All data was expressed as the relativeexpressed amount based on the expressed amount after 3 days in the caseof not loading the cell mass with BIO. Wnt10B is mainly expressed byepithelial cells during the stages of hair follicle morphogenesis andhair growth, and is known to be a gene involved in an epithelialcell-mesenchymal cell interaction. As shown in the drawing, in contrastto expression of Wnt10B only being observed to be expressed to about thesame level as that of a control cultured in the absence of BIO in cellsin which Wnt signaling was activated by culturing in the presence of BIOas long as the cells were cultured in the presence of BIO, expression ofWnt10B increased over time in cells in which cell differentiation wassubsequently induced by removing BIO. Accordingly, in this experimentalsystem, differentiation of follicular epithelial cells (outer rootsheath cells), and what is more, autonomous organogenesis were clearlydetermined to be induced by culturing in the presence of BIO followed byculturing after removing BIO.

Reactivity of Cell Mass to Hair Growth Stimulator, Cyclosporine A

Hypertrichosis is known to be a side effect of the immunosuppressant,cyclosporine A (Lutz, et al., Skin Pharmacol. 7, 101, 1994), andcyclosporine A has been clearly determined to demonstrate hair growthaction (Paus, et al., Lab. Invest. 60, 365, 1989) and hair growthpromoting action (Taylor, et al., J. Invest. Dermatol., 100, 237, 1993).

Hair papilla cells were prepared to P3 (with P1 indicating one round ofsubculturing) in the absence of BIO loading, while outer root sheathcells were prepared up to and including P3.

Each of the cells was diluted with each medium following trypsintreatment so as to obtain 3×10³ cells.

25 to 35 μl of each culture medium was dispensed onto the inside of theupper lid of a square culture dish measuring 10 cm on a side and mixedto prepare dome-shaped droplets of a suitable size.

After carefully closing the lid, the cells were cultured at 37° C. in a95% CO₂ atmosphere while ensuring that the culture medium did not dropdown.

Three days later, the medium was replaced with the same medium as thatused above containing cyclosporine A. Cyclosporine A was dissolved to 10mM with ethanol and added to a concentration of 0.1 to 10 μM.

The samples were collected three days after addition of cyclosporine Aand submitted for RT-PCR analysis (4) as described below.

RT-PCR Analysis (4)

RNA was extracted from the samples using the RNeasy Kit (Qiagen). Anequivalent of 200 ng of each RNA was reverse transcribed to cDNA with areaction system consisting of 20 μl of SuperScript II (Invitrogen). 1 μlof the resulting samples were applied to a quantitative PCR system(LightCycler System, Roche) in a 20 μl system using the primers listedbelow. The reaction protocol of LightCycler FastStart DNA Master SYBRGreen (Roche) (consisting of 40 cycles of 10 seconds at 95° C., 10seconds at 58° C. and 15 seconds at 72° C.) was used.

BMP4: Sense primer (forward chain): (SEQ ID NO. 17)gggcacctcatcacacgact  Anti-sense primer (reverse chain): (SEQ ID NO. 18)ggcccaattcccactccctt  c-myc: Sense primer (forward chain):(SEQ ID NO. 19) ttctctccgtcctcggaattctctg Anti-sense primer (reverse chain): (SEQ ID NO. 20)cagcagaaggtgatccagactctgac  IGFBP3: Sense primer (forward chain):(SEQ ID NO. 21) acagccagcgctacaaagtt  Anti-sense primer (reverse chain):(SEQ ID NO. 22) tagcagtgcacgtcctcctt 

The results are shown in FIG. 10. All data was expressed as the relativeexpressed amount based on the case of not adding Cyclosporine A. BMP4 isknown to be a gene that is mainly expressed by hair papilla cells duringthe hair follicle morphogenesis and hair growth stages and acts toinhibit epithelial cell-mesenchymal cell interaction (Hens, et al.,Development 134, 1221, 2007). As shown in the drawing, the expressedamount of BMP4 decreased significantly to about 60%. In addition, c-mycis known to be a gene that is strongly expressed in the bulge regionduring the hair development stage and in hair matrix cells during thegrowth stage, and acts positively on the proliferation of follicularepithelial cells (Bull, J. J. et al., Invest. Dermatol. 116, 617, 2001).As shown in the drawing, expression of c-myc increased to nearly 1.5times that of the control. Moreover, IGFBP3 is known to be factor thatinhibits hair growth (Weger, et al., J. Invest. Dermatol. 125, 847,2005), and the expression thereof has been clearly demonstrated toincrease during the dormant stage (Schlake, et al., Gene Expr. Patterns4, 141, 2004). As shown in the drawing, expression of IGFBP3significantly decreased to about 60%. Accordingly, genes involved inhair development and hair growth used in this experimental system weredetermined to be altered by Cyclosporine A, thereby demonstrating thatthis experimental system can be used to evaluate the effects of drugs onhair development and hair growth.

The invention claimed is:
 1. A method of producing a cell mass capableof serving as a primitive organ-like structure comprised of combinationsof outer root sheath (ORS) cells and mesenchymal somatic cells,comprising: preparing cultures containing ORS cells and mesenchymalsomatic cells; mixing said ORS cells and mesenchymal somatic cellscultures followed by adding a Wnt signal activator which inhibitsglycogen synthase-kinase-3 (GSK-3) to the mixed cell culture; subjectingthe mixed cell culture containing the Wnt signal activator to a hangingdrop method or spheroid formation method over a predetermined timeperiod; and replacing the medium of the culture cultured by the hangingdrop method or spheroid formation method with medium not containing Wntsignal activator and further culturing for a predetermined time period;wherein the mesenchymal somatic cells are maintained in anundifferentiated state, wherein the ORS cells are maintained in anormal, differentiated state and wherein the mesenchymal somatic cellsare hair papilla cells and the ORS cells are keratinocytes.
 2. Themethod according to claim 1, wherein hair follicles having a hairfollicle inducing function are formed from the cell mass.
 3. The methodaccording to claim 1, wherein the Wnt signal activator is6-bromoindirubin-3′-oxime (BIO).
 4. The method according to claim 1,wherein the Wnt signal activator demonstrating an inhibitory activity onGSK-3 is selected from bis-indolo(indirubin) compound,(2′Z,3′E)-6-bromoindirubin-3′-oxime,(2′Z,3′E)-6-bromoindirubin-3′-acetoxime,4-benzyl-2-ethyl-1,2,4-thiadiazolidine-3,5-dione,2,4-dibenzyl-5-oxothiazolidine-3-thione,2-chloro-1-(4,4-dibromo-thiophen-2-yl)-ethanone,[alpha]-4-dibromoacetophenone,N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea) and aGSK-3[beta]peptide inhibitor.